Problem…the immune system makes over one billion different antibody proteins • In 1950’s: central dogma stated DNA—to RNA—to protein • One gene for each protein • Required millions of genes just for the immune system • Does not seem possible, but most scientists thought it might be • Today we know the human genome is less than 30,000 genes • So, what is really going on???
Current theory must account for the following known properties of antibodies
Germ-line vs somatic-variation theories • Germ-line: stated that each antibody had its own gene….nothing special, but required billions of genes to account for numbers of antibodies • Somatic-variation: some mutation and recombination created vast number of genes for antibody formation • This introduced a new concept: targeted mutation or recombination of DNA: is it possible?? • Paradox: how could stability be maintained in C region and diversity exist in V region?
Dreyer and Bennett • In 1965 proposed radical theory to account for diversity of antibodies • Each antibody was coded for by two separate genes • One for the variable region • One for the constant region • Combined at the DNA level and expressed single mRNA • Suggested 1000’s of variable region genes and only one constant region gene • Close, but no cigar • Most biomedical scientists did not like this idea and rejected it
Tonegawa’s demonstration • 1976—used restriction enzymes and DNA probes to show that germ cell DNA contained several smaller DNA segments compared to DNA taken from developed lymphocytes (myeloma cells)
Mechanism of variable region rearrangements • Each V, D and J segments of DNA are flanked by special sequences (RSS—recombination signal sequences) of two sizes • Single turn and double turn sequences (each turn of DNA is 10 base pairs long) • Only single turn can combine with a double turn sequence • Joining rule ensures that V segment joins only with a J segment in the proper order • Recombinases join segments together
P and N region nucleotide alteration adds to diversity of V region • During recombination some nucleotide bases are cut from or add to the coding regions (p nucleotides) • Up to 15 or so randomly inserted nucleotide bases are added at the cut sites of the V, D and J regions (n nucleotides_ • TdT (terminal deoxynucleotidyl transferase) a unique enzyme found only in lymphocytes • Since these bases are random, the amino acid sequence generated by these bases will also be random
Randomness in joining process helps generate diversity by creating hypervariable of antigen binding site
Some rearrangements are productive, others are non-productive: frame shift alterations are non-productive
Somatic hypermutation adds even more variability • B cell multiplication introduces additional opportunities for alterations to rearranged VJ or VDJ segments • These regions are extremely susceptible to mutation compared to “regular” DNA, about one base in 600 is altered per two generations of dividing (expanding) lymphocyte population
Combination of heavy and light chains adds final diversity of variable region • 8262 possible heavy chain combinations • 320 light chain combinations • Over 2 million combinations • P and N nucleotide additions and subtractions multiply this by 104 • Possible combinations over 1010
Location of variability occurs within CDR regions of V domains (antigen binding sites)
Class switching among constant regions: generation of IgG, IgA and IgE with same antigenic determinants—idiotypes
Regulation of Ig gene transcription • Each lymphocyte rearranged gene has regulatory sequences that control gene expression • Promoters: initiation sites of RNA transcription • Enhancers: upstream of downstream that transcription from the promoter sequence • Silencers: down-regulate transcription in germline cells • Gene rearrangement brings enhancer and promoter regions close together and eliminates silencer regions allowing transcription
Understanding of immunoglobulin structure and formation has opened up a new world of possibilities • Monoclonal antibodies • Engineering mice with human immune systems • Generating chimeric and hybrid antibodies for clinical use • Abzymes: antibodies with enzyme capability